Proximity-based labeling of sialylated glycoproteins

Abstract

Described herein are systems and methods capable of analyzing localized microenvironments across sialylated proteomes by proximity markers. In one aspect, described herein are conjugates having compositions and electronic structures for producing reactive labeling intermediates in the microenvironment of sialylated cell surface glycoproteins. In some embodiments, the conjugate comprises a transition metal catalyst coupled to a cell surface glycoprotein. As further described herein, the transition metal catalyst may be coupled to a glycoprotein via a derived sialic acid linker.

Description

[0001] Related Application Data

[0002] Under Article 8 of the Patent Cooperation Treaty, this application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 419,519, filed on Oct. 26, 2022, which is hereby incorporated by reference in its entirety. Field of the Invention

[0003] The present invention relates to proximity-based labeling compositions, systems, and methods, particularly proximity-based labeling to profile local microenvironments across the sialylated proteome. Background Art

[0004] Glycosylation is one of the most common post-translational modifications (PTMs) on proteins, occurring on at least 50% of all known mammalian proteins and significantly increasing the functional proteome. Glycosylation can alter protein localization and function, and misregulated deposition has been shown to contribute to various disease phenotypes such as cancer metastasis, viral immune escape, viral entry, and inflammation. Glycoproteins also play a key role in the entire cell surface architecture, thus contributing to cell adhesion, cell signaling, viral docking, and cell-cell interactions. Among the monosaccharide arrays on the cell surface, sialic acid has a particularly strong impact on cell function. This charged sugar is incorporated by sialyltransferases and typically modifies the ends of polysaccharide chains. During tumor formation, overexpression of sialyltransferases leads to hypersialylation, which in turn promotes tumor progression through two different paradigms: (1) sialylation appears to inhibit apoptosis and allow cells to evade the immune system, and (2) the sialoglycoconjugate sialyl Lewis X (sialylLewis X ) promotes metastasis by extravasating cancer cells from the bloodstream into nearby tissues.

[0005] Despite these observations, the underlying biochemical mechanisms are still poorly understood, in part due to the lack of high-resolution tools to evaluate the functional role of sialylation. Summary of the Invention

[0006] In view of the foregoing drawbacks, the present disclosure describes systems and methods for interrogating the local microenvironment across the sialylated proteome by proximity labeling. In one aspect, the present disclosure describes conjugates having compositions and electronic structures for generating reactive labeling intermediates in the microenvironment of sialylated cell surface glycoproteins. In some embodiments, the conjugate comprises a transition metal catalyst coupled to a cell surface glycoprotein. As further described herein, the transition metal catalyst may be coupled to the glycoprotein via a derivatized sialic acid linker. In some embodiments, the transition metal catalyst and the derivatized sialic acid linker are coupled by click chemistry. Suitable click chemistry moieties for the transition metal complex and / or the derivatized sialic acid linker may be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide. For example, sialic acid may be derivatized to include a suitable click chemistry moiety for coupling to the transition metal catalyst. Figure 1 Illustrated is the use of sialic acid derivatized with an azide functional group for reaction with the DBCO moiety of a transition metal catalyst. In some embodiments, the derivatized sialic acid linker is incorporated into the cell surface glycoprotein by metabolism, followed by reaction to capture the transition metal catalyst. Figure 1 Illustrated are non-limiting embodiments in which an azide-derivatized sialic acid linker is incorporated into the cell surface glycoprotein by metabolism. In some embodiments, the transition metal catalyst may comprise a platinum group metal center. Additionally, in some embodiments, the transition metal catalyst is of Formula I:

[0007]

[0008] wherein M is a transition metal;

[0009] wherein A, D, E, G, Y, and Z are independently selected from C and N;

[0010] wherein R 3 to R 7 each represent one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 , and -R 9 OH, wherein R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl;

[0011] wherein R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocycloalkylene, and heteroarylene;

[0012] wherein L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; and

[0013] R 2 is selected from the group consisting of alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic group, hydroxyl, carboxyl, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 、-OS(O2)R 9 、thiol, biotin, oxyamine, and haloalkyl, wherein R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl ester; and wherein X - is a counterion, and n is an integer from 0 to 20. As provided in Formula I, the linking moiety L is optional, and thus L may be absent in some embodiments of the transition metal catalyst.

[0014] By selecting R 3 to R 7 the polarity of the transition metal complex can be adapted to a specific cellular environment. In some embodiments, for example, selecting one or more of R 3 to R 7 exhibits hydrophilic characteristics through charged and / or polar chemical moieties. In such embodiments, the transition metal complex can exhibit hydrophilic characteristics suitable for placement in the intercellular / extracellular environment. For example, Figure 6 the transition metal complex shown incorporates charged and polar chemical moieties for an aqueous intercellular environment. Alternatively, selecting one or more of R 3 to R 7 exhibits hydrophobic, lipophilic, or nonpolar characteristics.

[0015] Transition metal catalysts can have an electronic structure for transferring energy to a protein labeling reagent to generate a reactive intermediate. In some embodiments, the energy transfer is Dexter energy transfer or electron transfer. In some embodiments, the energy transfer to the protein labeling reagent can originate from an excited state of the transition metal catalyst's electronic structure. The excited state of the catalyst can be, for example, a singlet excited state or a triplet excited state. The excited state of the catalyst can be generated by one or more mechanisms, including energy absorption by the catalyst. In some embodiments, the catalyst is a photocatalyst, where the excited state is induced by absorbing one or more photons. In other embodiments, the catalyst can be in an excited state by interacting with one or more chemical species in the surrounding environment. Alternatively, the energy transfer (including electron transfer) to the protein labeling reagent can originate from the ground state of the catalyst's electronic structure.

[0016] In another aspect, the present disclosure provides a system for analyzing the local microenvironment of a sialylated proteome. In some embodiments, the system includes a protein labeling reagent and a conjugate that includes a transition metal catalyst coupled to a cell surface glycoprotein via a derivatized sialic acid linker, where the transition metal catalyst has an electronic structure that permits energy transfer to the protein labeling reagent to provide a reactive intermediate. The reactive intermediate is operative to label proteins or other biomolecules within a predetermined radius of the conjugate. The predetermined radius can be the diffusion radius of the reactive intermediate.

[0017] The diffusion radius of the reactive intermediate can be adapted for specific microenvironment mapping (based on proximity labeling) considerations and can be restricted to the nanoscale. In some embodiments, for example, the diffusion radius of the reactive intermediate can be less than 100 nm, less than 50 nm, less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm before quenching in the surrounding environment. In some embodiments, the diffusion radius can be from 0.5 nm to 10 nm. Thus, the reactive intermediate will react or crosslink with proteins or other biomolecules within the diffusion radius, or if no proteins or biomolecules are present, the reactive intermediate is quenched by the surrounding environment. In this way, a high-resolution local environment can be mapped through the concerted effort between the catalyst and the protein labeling reagent. Additionally, in some embodiments, the reactive intermediate can exhibit a t of less than 5 ns, less than 4 ns, or less than 2 ns before quenching. 1 / 2 . For example, the reactive intermediate can exhibit a t of less than 1 to 5 ns. 1 / 2 . In additional embodiments, by extending the half-life of the reactive intermediate, the diffusion radius can be extended to between 5 and 500 nm. For example, in some embodiments, the half-life of the reactive intermediate can be 1 to 100 μs or longer.

[0018] In some embodiments, the protein labeling reagent can be diazirine. Triplet energy transfer from an excited state photocatalyst can promote diazirine to its triplet state (T1). The diazirine triplet undergoes N2 elimination to release a free triplet carbene, and the free triplet carbene undergoes picosecond timescale spin equilibration to its reactive singlet state (t 1 / 2 <1 ns), which crosslinks with nearby proteins or is quenched in an aqueous environment. In some embodiments, the extinction coefficient of the transition metal complex is 3 to 5 orders of magnitude greater than that of diazirine.

[0019] Any diazirine conforms to the technical principles discussed herein. For example, diazirine sensitization can be extended to various para- and meta-substituted aryl trifluoromethyl diazirines with valuable payloads for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. Diazirines can be functionalized with markers such as biotin. In some embodiments, the marker is desthiobiotin. The marker can help identify the protein labeled by the protein labeling reagent. For example, the marker can be used for assays by protein immunoblot (western blot) and / or other analytical techniques. In addition to biotin and desthiobiotin, markers can include alkyne, azide, FLAG tag, fluorophore, and chloroalkane functional groups.

[0020] In additional embodiments, where the transition metal catalyst is a photocatalyst, the protein labeling reagent can be an azide. Triplet energy transfer from an excited state photocatalyst can promote the formation of nitrene from the azide. The reactive nitrene crosslinks with nearby proteins or is quenched in an aqueous environment. Any azide operable to undergo energy transfer with the transition metal photocatalyst for the formation of nitrene can be employed. In some embodiments, the azide is an aryl azide.

[0021] In another aspect, methods are provided for analyzing the local microenvironment of sialylated proteomes. The methods include: forming a conjugate comprising a transition metal catalyst coupled to a cell surface glycoprotein via a derivatized sialic acid linker; and using the transition metal catalyst to activate a protein labeling reagent to a reactive intermediate. The reactive intermediate couples to a protein or other biomolecule within a predetermined radius of the conjugate. The transition metal catalyst, protein labeling reagent, and reactive intermediate can have any composition and / or properties described herein.

[0022] These and other embodiments are further described in the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shows microenvironment mapping using the system described herein, which comprises a conjugate and a protein labeling reagent, the conjugate comprising a transition metal catalyst coupled to a cell surface glycoprotein via a derivatized sialic acid linker, wherein the transition metal catalyst has an electronic structure that permits energy transfer to the protein labeling reagent to provide a reactive intermediate.

[0024] Figure 2A Shows the workflow of the glycomap experiment. The Ac4ManNAz incubation was carried out for 72 h, however, shorter times in the cell lines used in this study resulted in equivalent results. Optimization of the Ir-DBCO incubation time is shown in the accompanying Supplementary Information (Figure S3).

[0025] Figure 2B Provides Western blot analysis of proteins in whole cell lysates after the glycomap experiment.

[0026] Figure 2C Provides immunofluorescence analysis of cells after the glycomap experiment; Red: streptavidin, Blue: Hoechst.

[0027] Figure 2D Is a Western blot of streptavidin-enriched lysates stained for Nicastrin (top lane) and CD55 (bottom lane).

[0028] Figure 3A Shows the workflow of TMT-based chemical proteomic discovery of the interactome of sialylated glycoproteins. Each experiment was repeated three times.

[0029] Figure 3B Provides quantitative chemical proteomic validation of glycomap in HEK293T cells. For all experiments, the same cut-off values (>1.5 Log2 (fold change); >1.5 - Log 10 (p-value)) were used.

[0030] Figure 4A Summarizes comparative proteomic experiments of primary cervical cells (PCC) and HeLa cells. Top row: sialylated glycoproteins in PCC (left) and HeLa (right). Bottom row: interacting proteins in PCC (left) and HeLa cells (right). Middle: Venn diagram of enriched proteins from each dataset. The same cut-off values (>1.5 Log2 (fold change); >1.5 - Log 10 (p-value)) were used for the analysis of all datasets.

[0031] Figure 4BGene Ontology (GO) analysis of the identified sialylated glycoproteins (upper panel) and their interactors (lower panel).

[0032] Figure 4C Venn diagram of enriched solute carrier proteins (SLCs) that interact with sialylated glycoproteins.

[0033] Figure 5A Shows the workflow of metabolomics analysis of HeLa cells.

[0034] Figure 5B Metabolite levels of selected small molecules were quantified. The experiment was repeated three times.

[0035] Figure 5C - Left: GO analysis indicates that cation homeostasis is affected by sialylation. Middle: Zinc transporters enriched in the HeLa and PCC datasets. Right: Colorimetric zinc assay, which shows a significant change in cellular zinc levels in response to desialylation. P values determined by unpaired Student's t-test. *P < 0.05, **P < 0.01.

[0036] Figure 6 Shows a transition metal complex incorporating charged and / or polar chemical moieties for an aqueous intercellular environment according to some embodiments.

[0037] Figure 7 Shows various chemical substances employed in some embodiments of the compositions and methods described herein. Detailed Description

[0038] The embodiments described herein can be more readily understood by reference to the following detailed description and examples, as well as the description before and after them. However, the elements, devices, and methods described herein are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the invention. Many modifications and adaptations will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

[0039] Definition

[0040] The term "alkyl" as used herein alone or in combination refers to a straight-chain or branched-chain saturated hydrocarbon group optionally substituted with one or more substituents. For example, alkyl can be C1-C 30 or C1-C 18 .

[0041] As used herein, the term "alkenyl", alone or in combination, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.

[0042] As used herein, the term "alkynyl", alone or in combination, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond and optionally substituted with one or more substituents.

[0043] As used herein, the term "aryl", alone or in combination, refers to an aromatic monocyclic or polycyclic system optionally substituted with one or more ring substituents.

[0044] As used herein, the term "heteroaryl", alone or in combination, refers to an aromatic monocyclic or polycyclic system in which one or more ring atoms are elements other than carbon (e.g., nitrogen, boron, oxygen, and / or sulfur).

[0045] As used herein, the term "heterocycle", alone or in combination, refers to a monocyclic or polycyclic system in which one or more atoms of the ring system are elements other than carbon (e.g., boron, nitrogen, oxygen, and / or sulfur or phosphorus) and in which the ring system is optionally substituted with one or more ring substituents. The heterocyclic system may include aromatic and / or non-aromatic rings, including rings having one or more sites of unsaturation.

[0046] As used herein, the term "cycloalkyl", alone or in combination, refers to a non-aromatic monocyclic or polycyclic system optionally substituted with one or more ring substituents.

[0047] As used herein, the term "heterocycloalkyl", alone or in combination, refers to a non-aromatic monocyclic or polycyclic system in which one or more atoms of the ring system are elements other than carbon (e.g., boron, nitrogen, oxygen, sulfur, or phosphorus (alone or in combination)) and in which the ring system is optionally substituted with one or more ring substituents.

[0048] As used herein, the term "alkoxy" refers to the moiety RO—, where R is an alkyl, alkenyl, or aryl as defined above.

[0049] As used herein, the term "halogen" refers to an element of Group VIIA or Group 17 of the periodic table (halogens). Depending on the chemical environment, the halogen may be in a neutral or anionic state.

[0050] Terms not specifically defined herein are given their ordinary meaning in the art.

[0051] Embodiments of the present application are further illustrated in the following non-limiting examples.

[0052] An iridium photocatalyst was developed for microenvironment mapping to reveal the interactome of sialylated cell surface glycoproteins, as Figure 1 illustrated. According to Figure 2AResearch using iridium photocatalysts was initiated. HeLa cells were incubated with tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz) and then treated with DBCO-iridium ( Figure 7 , S1), resulting in the incorporation of the iridium photocatalyst onto glycoproteins. Irradiation in the presence of biotin-diazirine ( Figure 7 , S2) led to cell surface biotinylation, as observed by western blotting. ( Figure 2B ). Control reactions showed minimal biotinylation when the azidosugar, the DBCO-iridium reagent, or blue light irradiation was omitted. Importantly, immunoprecipitation on streptavidin beads showed strong enrichment of the known sialylated glycoproteins nicastrin (NCSTN) and complement decay-accelerating factor (CD55), giving confidence to our workflow ( Figure 2C ).

[0053] This method was compared to the direct biotinylation of Ac4ManNAz using DBCO-biotin (S3), and labeling of the sialome was observed by western blotting. These results indicate that our catalytic labeling method can install approximately 0.7 tags per catalyst (see ESI), representing a significant improvement over existing PAL probes, which typically react with water (>95%) and show minimal protein labeling. Finally, this protocol was also applicable to HEK293T cells and primary cervical cells (PCC) ( Figure 2D ), thus highlighting the versatility of the workflow. In all cases, confocal microscopy revealed strong biotinylation after treatment with DBCO-iridium or DBCO-biotin ( Figure 7 , S3) and irradiation in the presence of biotin-diazirine. No labeling was observed in any case in the absence of the azidosugar.

[0054] Encouraged by these results, a tandem mass tag (TMT)-based quantitative chemical proteomics workflow was developed to identify sialylated cell surface glycoproteins and map their interactomes ( Figure 3A)。For each cell type, we performed comparative experiments using three different conditions: The first condition (Condition A) involved SPAAC using DBCO-biotin, resulting in the biotinylation of only sialylated glycoproteins. Condition B utilized SPAAC using DBCO-iridium for the biotinylation of sialylated glycoproteins and their cognate interactomes via μMap. Finally, a control experiment was performed using DBCO-iridium in the absence of Ac4ManNAz. Together, these parameters allowed the identification of 1) sialylated glycoproteins, 2) sialylated glycoproteins and their local interactomes, and 3) protein interactors that selectively recognize the cell surface sialome (GlycoMap). We first examined this chemoproteomic workflow on HEK293T cells ( Figure 3B ). Using Condition A (vs. control), we found significant enrichment of 363 proteins (>1.5 Log2 (fold change); >1.5 - Log 10 (p-value)), 93% of which were known glycoproteins, including nicastrin (NCSTN), cadherin 2 (CDH2), small cell adhesion glycoprotein (SMAGP), cluster of differentiation 47 (CD47), basigin (BSG), cluster of differentiation 166 (CD166), cluster of differentiation 99 (CD99), and neuroplastin (NPTN). As Figure 3A predicted, Condition B (vs. control) enriched sialylated glycoproteins as well as proximal proteins, thus sharing approximately 65% overlap with the proteins enriched in Condition A (vs. control) (Figure S9). Analysis of the sialic acid interactome generated by GlycoMap showed that 81% of the enriched proteins were membrane-associated, thus reflecting the accuracy of our labeling method. Additionally, several lysosomal proteins (21) were enriched, presumably generated by the internalization of iridium-bound glycoproteins prior to proximity labeling.

[0055] In the initial analysis of the GlycoMap dataset, the known membrane-bound protein complex, γ-secretase, was examined to validate our method. This heterotetramer of membrane proteins (NCSTN, APH1A, PSEN1, PEN-2) proteolytically cleaves many integral membrane proteins, but only NCSTN is directly sialylated. In our dataset, NCSTN was highly enriched (3.8 log2FC) in Condition A (vs. control), while the non-sialylated interactor APH1A was strongly enriched (3.3 log2FC) in the GlycoMap arm, thus demonstrating that the μMap workflow can delineate the relationship between sialylated glycoproteins and their interactors.

[0056] A workflow has been established, and next, we seek to investigate the events of high sialylation in tumor formation. To examine this, we performed a GlycoMap experiment comparing primary cervical cancer cells (PCC) and the HeLa cervical adenocarcinoma cell line ( Figure 4A ).

[0057] Consistent with previous observations of upregulated sialylation, chemical proteomic analysis revealed significantly higher sialylation in HeLa cells (447 enriched proteins) compared to PCC (223 enriched proteins)( Figure 4A ). Thus, the increased sialoglycome in HeLa cells resulted in a higher number of interacting proteins (166 enriched proteins in HeLa cells vs 63 enriched proteins in PCC).

[0058] Next, we performed a global gene ontology (GO) analysis to classify the enriched sialylated proteins and their interactors ( Figure 4B ). The functional enrichment of both cell types was in good agreement with the following known roles of the identified glycoproteins: cell adhesion, host cell entry, and regulation of migration, death, and defense. In addition, GO terms with significant differences (≥10Log10(p-value) change) between primary and cancer cervical cells were examined, verifying that sialoglycoproteins in cancer cells are associated with typical tumor phenotypes, including cell morphogenesis, cell-cell adhesion, extracellular matrix organization, and tube morphogenesis.

[0059] Interestingly, when comparing the roles of the identified sialic acid-interacting proteins, terms related to small molecule transport were significantly enriched in HeLa cells (organic ion transport, transport of small molecules, vitamin transport), and we specifically noted the large enrichment of solute carrier proteins (SLC) in this dataset ( Figure 4C ). In particular, the interactions of sialylated proteins with SLCs related to ethanolamine, carnitine, and zinc transport were all significantly more enriched in HeLa cells than in PCC.

[0060] To explore the potential consequences of these interactions, we sought the metabolic consequences of their enzymatic depletion ( Figure 5A) Treatment of HeLa cells with sialidase (VC-Sia) isolated from Vibrio Cholerae effectively cleaves sialic acids linked α2,3-, α2,6-, or α2,8- to cell surface glycans, enabling us to modulate the overall sialylation status. We incubated HeLa cells in the presence or absence of VC-Sia and then performed mass spectrometry-based metabolomics quantification of cell metabolite extracts. Although most metabolite levels were minimally affected by sialidase treatment, we found that the levels of ethanolamine derivatives were significantly increased in sialidase-treated cells ( Figure 5B ), and the ethanolamine derivatives include cytidine diphosphate ethanolamine (CDP-Etn), phosphoethanolamine (P-Etn), and cytidine diphosphate choline (CDP-choline). The solute carrier protein responsible for ethanolamine transport, choline-like transporter 1 (SLC44A1), is not known to be glycosylated, but our dataset suggests that its function may be regulated by neighboring sialylated glycoproteins. Based on these results, we hypothesized that cell surface sialic acids may present a negatively charged surface around membrane-bound transporters and may thereby affect the transport of ions, including metabolites.

[0061] Similarly, we also investigated the effect of sialylation on zinc uptake ( Figure 5C ). Zinc enters through the cell membrane via a family of solute carrier proteins of the SLC39 family, four of which are shown to be sialylated in our HeLa dataset (SLC39A6, SLC39A8, SLC39A10, and SLC39A14), and one of which (SLC39A1) is thought to interact with sialylated glycoproteins. Zinc is a key micronutrient that plays an important role in cell function and whose transport is dysregulated in many cancers.

[0062] Using a colorimetric assay to measure the zinc levels in untreated and sialidase-treated HeLa cells, we found that the zinc levels were significantly higher in cells treated with VC-Sia. These data suggest that cell surface sialylation and / or interactions with sialylated glycoproteins play a role in the regulation of cellular zinc concentration.

[0063] In summary, high sialylation in cancer has recently attracted great interest in academic and pharmaceutical sectors. However, tools for understanding the biochemical consequences of high sialylation remain limited. Here, we describe a novel proximity labeling platform to identify sialylated cell surface glycoproteins and their interactors. This sensitive and precise method is robust and compatible with various cell lines, including primary cells. Our comparative proteomics study between primary and cancer cervical cell lines revealed a significant link between sialylation and solute carrier proteins, thus suggesting a new role for sialylation. Metabolomics data indicated that these interactions regulate the function of certain solute carriers. In summary, our platform represents a powerful new approach for sialic acid interactome analysis, thus providing a system-level tool for elucidating the biochemical consequences of high sialylation.

[0064] Material

[0065] All buffers and materials were obtained from commercial sources. N-Azidoacetyl-4,6-O-benzylidene-D-mannosamine (Ac4ManNAz) (900917), bovine serum albumin (BSA) (A7906), and Eppendorf Protein LoBind tubes (Z666505) were purchased from Millipore Sigma (St. Louis, MO, USA). DBCO-Sulfo-Link-Biotin (DBCO-Biotin) (BP-22296) was purchased from Broadpharm (San Diego, CA, USA). Biotin-(PEG)3-Diazirine (Biotin-Diazirine) and [Ir(dCO2HdFCF3ppy)2(bpy-dbco) (DBCO-Iridium) were synthesized as previously described. 1,2RIPA buffer (89900), 1X DPBS (14190144), Pierce BCA Protein Assay Kit (23227), and iBright Prestained Protein ladder (LC5615) were purchased from Thermo Scientific (Rockford, Illinois, USA). TBST (IBB-581X) was purchased from Boston BioProducts (Ashland, Massachusetts, USA). 12% Criterion TGX precast gels (5671044) and 4x Laemmli sample buffer (161-0747) were purchased from BioRad (Hercules, California, USA). Poly-L-lysine solution was obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Paraformaldehyde (16% solution) was obtained from Thermo Fisher Scientific (Rockford, Illinois, USA). Streptavidin-Alexa Fluor 488 was obtained from BioLegend (San Diego, California, USA). Standard tissue culture dishes were obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). DPBS (Gibco, #14190250), DMEM high glucose (Gibco, #31053036), DMEM high glucose - phenol red free (Gibco, #31053028), fetal bovine serum (Gibco, #10437-028), penicillin-streptomycin (Gibco, #15070063), trypsin-EDTA (Gibco, #25300054), trypsin protease MS (Pierce, #PI90057), and RIPA buffer (Thermo, #89900) were obtained from Thermo Fisher Scientific. PMSF (Sigma Aldrich, #78830) and complete EDTA-free protease inhibitor (Roche, #11873580001) were obtained from Sigma Aldrich. Streptavidin magnetic beads were obtained from Thermo Fisher Scientific (Pierce, #88816). Trifluoroacetic acid (Optima grade), acetonitrile (Optima grade), water (Optima grade), and acetic acid (Optima grade) were obtained from Thermo Fisher Scientific.Triethylammonium bicarbonate (1M Sigma Aldrich, #90360), 50% hydroxylamine solution (Sigma Aldrich, #438227), ammonium bicarbonate (LiChropur, Merck, #5438350) and iodoacetamide (Sigma Aldrich, #I1149) were obtained from Sigma Aldrich. TMT10plex kit (Thermo), urea (Pierce, Sequanal, #29700) and DTT (Thermo, #R0862) were obtained from Thermo Fischer Scientific.

[0066] Cell line

[0067] HEK293 (CRL321) and HeLa cells (CCL2) were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Gibco, #31053036) supplemented with 10% fetal bovine serum (Gibco, #10437-028) and 1% penicillin-streptomycin (Gibco, #15070063) in 10 cm culture dishes at 37 °C and 5% CO2 atmosphere.

[0068] Primary cervical epithelial cells (ATCC, #PCS-480-011) were cultured in the recommended medium (ATCC, #PCS-480-032) supplemented with the recommended growth kit (ATCC, #PCS-480-042) according to the protocol provided by ATCC.

[0069] Antibody

[0070] Anti-NCSTN (rabbit, polyclonal): Invitrogen (#PA5-17735).

[0071] Anti-CD55 (rabbit, polyclonal): Invitrogen (#PA5-29657).

[0072] Anti-actin (mouse, monoclonal): Cell Signaling Technology (#3700S).

[0073] Glycomap experiment

[0074] The general experimental workflow is shown in Figure 2A in.

[0075] Optimization of Glycomap conditions

[0076] HEK293T cells (approx. 0.4×10 6 cells) were incubated in 6-well culture dishes in complete DMEM (2 mL) at 37 °C for 72 h in the presence or absence of Ac4ManNAz (100 μM). Cells were washed with DPBS (3 × 1 mL), then incubated in complete DMEM (2 mL) containing DBCO-Iridium (2.5 to 10.0 μM) at 37 °C for 3 to 24 h. Cells were washed with DPBS (3 × 1 mL) and irradiated in a biophotoreactor (blue LED) at room temperature in DMEM without phenol red (200 μL) containing biotin-diazirine (250 μM) for 20 min. Cells were washed with DPBS (3 × 1 mL), scraped in DPBS (1 mL), and transferred to 1.5 mL Eppendorf tubes. Cells were pelleted at 400×G for 5 min and resuspended in Ripa lysis buffer (500 μL). Cells were lysed by sonication (bioruptor) at 4 °C for 10 min (20 cycles, on for 15 sec off for 15 sec at 100% power). Protein concentrations were normalized by BCA assay and lysates were analyzed by protein immunoblotting (10 μg protein per lane, 12% gel, 150 V). Gels were transferred to NC membranes by iBlot 2. After transfer, membranes were stained with total protein stain, washed with wash solution (3 × 5 sec), and imaged in the 700 nm channel by Li-Cor Odyssey Clx scanner. Membranes were then immersed in Odyssey blocking buffer (Li-Cor, 927-50000) and incubated at room temperature for 1 h. Blocking solution was gently poured out and 10 mL of fresh blocking buffer containing 0.5 μL of IRDye 800CW streptavidin (Li-Cor, 926-32230) was added. The mixture was shaken for 60 min. Buffer was gently poured out and membranes were washed with 1X TBST (4 × 5 min) and water (3 × 5 sec) before imaging in the 800 nm channel by Li-Cor Odyssey Clx scanner. Labeling efficiency was evaluated by densitometry by comparison with experiments performed in the absence of any Ac4ManNAz.

[0077] Protein immunoblot analysis

[0078] HeLa cells (approx. 0.4×10 6Cells (HEK293T, HeLa, or primary cervical epithelial cells) (approx. 2×10

[0079] Confocal microscopy

[0080] cells) were incubated in a 6-well culture dish in complete DMEM (2 mL) containing Ac4ManNAz (100 μM) for 72 h. The cells were washed with DPBS (3×1 mL), and then incubated in complete DMEM (2 mL) containing DBCO-Iridium (5 μM) or DBCO-Biotin (5 μM) at 37 °C for 24 h. The cells were washed with DPBS (3×1 mL), and irradiated in a biophotoreactor (blue LED) in DMEM (without phenol red, 200 μL) containing Biotin-Diazirine (250 μM) at room temperature for 20 min. The cells were washed with DPBS (3×1 mL), scraped in DPBS (1 mL), and transferred to a 1.5 mL Eppendorf tube. The cells were pelleted at 400×G for 5 min and resuspended in Ripa lysis buffer (500 μL). The cells were lysed by sonication (bioruptor) at 4 °C for 10 min (20 cycles, on for 15 sec off for 15 sec at 100% power). Protein concentrations were normalized by BCA assay, and lysates were analyzed by Western blot analysis (20 μg protein per lane, 12% gel, 150 V). The gel was transferred to an NC membrane by iBlot 2. After transfer, the membrane was immersed in Odyssey blocking buffer (Li-Cor, 927-50000) and incubated at room temperature for 1 h. The blocking buffer was replaced with fresh blocking buffer (10 mL) containing anti-actin antibody (10 μL), and the membrane was shaken for 1 h. The buffer was gently poured out, and the membrane was washed with 1X TBST (4×5 min) and water (3×5 sec). The blocking solution was gently poured out, and 10 mL of fresh blocking buffer containing 1 μL of IRDye 680RD goat anti-mouse IgG secondary antibody (Li-Cor, 926-68070) and 1 μL of IRDye 800CW streptavidin (Li-Cor, 926-32230) was added. The mixture was shaken for 60 min. The buffer was gently poured out, and the membrane was washed with 1X TBST (4×5 min) and water (3×5 sec) before imaging in the 700 nm and 800 nm channels by a Li-Cor Odyssey Clx scanner. 4HeLa cells (approx. 5×10cells) were incubated in a polylysine-coated 8-well chamber slide in complete DMEM (200 mL) containing Ac4ManNAz (100 μM) at 37 °C for 48 h. The cells were washed with DPBS (3×200 μL), and then incubated in complete DMEM (200 μL) containing DBCO-Iridium (5 μM) or DBCO-Biotin (5 μM) at 37 °C for 24 h. The cells were washed with DPBS (3×200 μL), and irradiated in a bioreactor (blue LED) at room temperature in DMEM (without phenol red, 200 μL) containing biotin-diazirine (250 μM) for 20 min. The cells were washed with DPBS (3×200 μL), and fixed with pre-warmed 4% paraformaldehyde (200 μL) at room temperature for 30 min. The cells were washed with DPBS (2×200 μL), and blocked with 3% BSA in DPBS (200 μL) at room temperature for 1 h. The blocking buffer was replaced with fresh 3% BSA in DPBS (200 μL) containing Hoechst (1:1000) and streptavidin-AlexaFluor 555 conjugate (1:1000). The cells were stained at room temperature in the dark for 1 h, and then stored at 4 °C in the dark. The cells were imaged at 20× magnification on a NIKON A1R-SI microscope (Nikon Instruments, Melville, NY, USA). The images were processed with Fiji-ImageJ. The images shown represent multiple cross-sectional images taken during each period.

[0081] Streptavidin immunoprecipitation

[0082] 6Cells (number not specified) were incubated in a 10 cm culture dish in complete DMEM (10 mL) containing Ac4ManNAz (100 μM) at 37 °C for 72 h. The cells were washed with DPBS (3 × 5 mL), then incubated in complete DMEM (5 mL) containing DBCO-Iridium (5 μM) at 37 °C for 24 h. The cells were washed with DPBS (3 × 5 mL) and irradiated in a bioreactor (blue LED) in DMEM without phenol red (5 mL) containing biotin-diazirine (250 μM) at room temperature for 20 min. The cells were washed with DPBS (3 × 5 mL), scraped in DPBS (5 mL), and transferred to a 15 mL conical tube. The cells were pelleted at 400×G for 5 min and resuspended in Ripa lysis buffer (1 μL) containing protease inhibitor cocktail. The cells were lysed by sonication (bioruptor) at 4 °C for 10 min (20 cycles, on for 15 sec and off for 15 sec at 100% power).

[0083] Protein concentrations were normalized by BCA assay. Lysates (0.75 mg protein / experiment) were added to Pierce Streptavidin beads (80 μL), and the beads were inverted at 4 °C for 16 h. The beads were washed with 1% SDS (3 × 500 μL, 5 min per wash), 1 M NaCl (3 × 500 μL), and 10% EtOH (3 × 500 μL). Proteins were then eluted with elution buffer / laemmli (3:1, 40 μL) and boiled at 95 °C for 15 min. The mother liquor was thermally separated and analyzed by protein immunoblotting and compared with the lysate input (10 μg protein per input lane) (12%, 150 V).

[0084] Transfer the gel to an NC membrane using iBlot 2. After transfer, immerse the membrane in Odyssey blocking buffer (Li-Cor, 927-50000) and incubate at room temperature for 1 hour. Replace the blocking buffer with fresh blocking buffer (10 mL) containing anti-CD55 antibody (10 μL), and shake the membrane at 4 °C for 16 hours. Gently pour out the buffer, and wash the membrane with 1X TBST (4×5 min) and water (3×5 sec). Gently pour out the blocking solution, and add 10 mL of fresh blocking buffer containing 1 μL of IRDye 800CW goat anti-rabbit IgG secondary antibody (Li-Cor, 926-32211). Shake the mixture at room temperature for 60 minutes. Gently pour out the buffer, and wash the membrane with 1X TBST (4×5 min) and water (3×5 sec) before imaging in the 800 nm channel using a Li-Cor Odyssey Clx scanner.

[0085] For subsequent staining, strip the membrane at room temperature for 30 minutes using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, 46430). Block and stain the membrane with anti-NCSTN antibody (1:1000) as described above.

[0086] Proteomics workflow

[0087] Seed HeLa cells (approx. 5×10 6 cells) in a 10 cm culture dish in complete DMEM (10 mL) containing Ac4ManNAz (100 μM) and incubate at 37 °C for 72 hours. Wash the cells with DPBS (3×5 mL), then incubate in complete DMEM (5 mL) containing DBCO-iridium (5 μM) at 37 °C for 24 hours. Wash the cells with DPBS (3×5 mL), and irradiate in a bioreactor (blue LED) in DMEM (without phenol red, 5 mL) containing biotin-diazirine (250 μM) at room temperature for 20 minutes. Wash the cells with DPBS (3×5 mL), scrape in DPBS (5 mL), and transfer to a 15 mL conical tube. Pellet the cells at 400×G for 5 minutes, and resuspend in Ripa lysis buffer (1 mL) containing protease inhibitor cocktail. Lyse the cells by sonication (bioruptor) at 4 °C for 10 minutes (20 cycles, on for 15 sec off for 15 sec at 100% power).

[0088] Normalize the protein concentration by BCA assay. Add the lysate (2.0 mg protein / experiment) to Pierce streptavidin beads (200 μL), and invert the beads at 4 °C for 16 h. Wash the beads with 1% SDS (3 × 500 μL, 5 min per wash), 1 M NaCl (3 × 500 μL), and 10% EtOH (3 × 500 μL). Resuspend the beads in RIPA buffer (500 μL), and transfer to a new 1.5 mL Lo-bind tube.

[0089] Remove the supernatant, and wash the beads with DPBS (3 × 500 μL) and NH4HCO3 (100 mM) (3 × 500 μL). Resuspend the beads in 6 M urea in DPBS (500 μL) and add 200 mM DTT in 25 mM NH4HCO3 (25 μL). Invert the beads at 55 °C for 30 min. Subsequently, add 500 mM iodoacetamide in 25 mM NH4HCO3 (30 μL), and invert the beads in the dark at room temperature for 30 min. Remove the supernatant, and wash the beads with DPBS (3 × 500 μL) and TEAB (50 mM) (3 × 500 μL). Resuspend the beads in TEAB (500 μL), transfer to a new protein LoBind tube, pellet, and remove the supernatant.

[0090] Resuspend the beads in 50 mM TEAB (40 μL), and add trypsin (1 mg / mL, in 50 mM acetic acid; 1.2 μL), and invert the beads at 37 °C overnight. After 16 h, add additional trypsin (0.8 μL), and invert the beads at 37 °C for an additional 1 h. Subsequently, pellet the beads. Meanwhile, equilibrate the TMT10 plex labeling reagent (0.8 mg) (Thermo) to room temperature, dilute with anhydrous acetonitrile (Optima grade; 41 μL, vortex for 5 min), and centrifuge to collect the contents.

[0091] Then add each set of trypsinized peptides to the corresponding TMT label (add 40 μL in TEAB to 41 μL in MeCN). Then wash the beads with additional TEAB (20 μL) to collect the remaining peptides. Allow the labeling reaction to proceed at room temperature for 2 h. Then quench the samples with 5% hydroxylamine (8 μL), and incubate at room temperature for 15 min. Concentrate the samples in a new protein LoBind tube, and quench with TFA (16 μL, Optima). Store the samples at -80 °C until proteomics is performed. Desalt and fractionate the samples before running.

[0092] Labeling efficiency

[0093] Based on Figure 2B the protein immunoblot results shown in, the labeling efficiency via Glycomap can be calculated using densitometry. In this calculation, it is assumed that the SPAAC efficiency of DBCO-iridium is comparable to that of DBCO-biotin. Using densitometry (MW range from 45 kDa to the top of the blot) in protein immunoblots stained with streptavidin, we calculated approximately 1 tag per catalyst.

[0094] The full calculation is as follows:

[0095] Densitometry reading for DBCO-iridium full experiment: 108,442

[0096] Densitometry reading for DBCO-iridium control: 6,164

[0097] Experiment / Control: 17.6

[0098] Densitometry reading for DBCO-biotin full experiment: 158,335

[0099] Densitometry reading for DBCO-biotin control: 5,937

[0100] Experiment / Control: 26.7

[0101] Comparison: 17.6 / 26.7 = 0.7 tags per catalyst.

[0102] Metabolomics experiment

[0103] Metabolite extraction

[0104] In transparent 10 cm culture dishes (x6), HeLa cells were cultured in 5 mL of complete DMEM supplemented with 10% FBS in the presence or absence of neuraminidase (20 U / mL) for 72 hours. After 72 hours, the cells were washed with DPBS (3 x 5 mL) and incubated with TrypLE (1 mL) at 37 °C for 15 minutes. The cells were transferred to a 15 mL conical tube with 4 mL of DPBS to wash the plate. The cells were pelleted at 500 x G for 4 minutes and resuspended in 1 mL of DPBS. The cells were counted and 500,000 cells / experiment were transferred to 1.5 mL Eppendorf tubes. The cells were pelleted (500 x g for 4 minutes), the supernatant was removed, and the cells were lysed with ice-cold 80% MeOH (60 μL) at 0 °C for 30 minutes. The cell lysate was clarified by centrifugation at 20,000 x G for 25 minutes, and the lysate was transferred to a new 0.5 mL Eppendorf tube and stored at -80 °C until mass spectrometry analysis.

[0105] LC-MS

[0106] HPLC-grade water, methanol, and acetonitrile were obtained from Thermo Fisher Scientific. The supernatant samples were thawed at room temperature and stored in the autosampler at 4 °C. The samples were analyzed using a Q Exactive Plus mass spectrometer coupled to a Vanquish UHPLC system (ThermoFisher Scientific). LC separation was achieved using an XBridge BEH Amide column (2.1 mm x 150 mm, 2.5 μm particle size, pore size; Milford, Massachusetts, USA, Waters) with a gradient of solvent A (20 mM ammonium acetate + 20 mM ammonium hydroxide in 95:5 water / acetonitrile [pH 9.45]) and solvent B (acetonitrile). The flow rate was 150 μL / min. The gradient was 0 min, 90% B; 2 min, 90% B; 3 min, 75%; 7 min, 75% B; 8 min, 70%; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 14 min, 25% B; 16 min, 0% B; 20.5 min, 0% B; 21 min, 90% B; 25 min, 90% B. The column temperature was 25 °C and the injection volume was 5 μL. The mass spectrometer was operated in full scan mode, running in positive and negative modes covering m / z 70 - 1000, with a resolution of 140,000 at m / z 200, AGC target 5E6, and a maximum injection time of 200 ms.

[0107] Data analysis

[0108] Data analysis was performed using El-Maven, and metabolites were identified through an in-house library using authentic standards. 3 Metabolite signal intensities were then further processed in Excel (median correction) and Graphpad Prism (transformation, t-test, and volcano plot generation).

[0109] Colorimetric Zinc assay

[0110] Zinc assays were purchased from Abcam (ab102507), and experiments were conducted according to the following protocol, which was adapted from the supplier's instructions.

[0111] In a transparent 10 cm culture dish, HeLa cells were cultured in complete medium (DMEM supplemented with 10% FBS) in the presence (x3) or absence (x3) of VC-sialidase (20 mU / mL medium) for 96 hours. The medium was changed every 24 hours, and fresh sialidase was added at these time points. After 96 hours, the medium was removed and DPBS (5 mL) was added. The cells were scraped, transferred to a 15 mL conical tube, and pelleted at 400×g for 5 minutes. The supernatant was removed, and the cells were lysed by sonication (bioruptor) in 50 μL of EDTA-free lysis buffer at 4°C for 10 minutes.

[0112] The lysate was clarified at 18,000×g for 15 minutes and analyzed by BCA assay. 30 μL of cell lysate was transferred to a 1.5 mL Eppendorf tube containing 30 μL of 7% TCA to precipitate proteins. The mixture was clarified at 18,000×g for 5 minutes, and 50 μL of the resulting solution was used for zinc detection assay.

[0113] Zinc concentration (nmol zinc / mg protein-free lysate):

[0114] 1 (untreated): 1.7 nmol / mg

[0115] 2 (untreated): 2.0 nmol / mg

[0116] 3 (untreated): 1.8 nmol / mg

[0117] 4 (Sia-treated): 1.4 nmol / mg

[0118] 5 (Sia-treated): 1.4 nmol / mg

[0119] 6 (Sia-treated): 1.4 nmol / mg

[0120] Comparison of zinc levels in PCC and HeLa cells

[0121] In a transparent 10 cm culture dish, HeLa cells (x3) and PCC cells (x3) were cultured in complete medium for primary cervical cells (ATCC, PCS-480-032) for 24 hours. The medium was removed and DPBS (5 mL) was added. The cells were scraped, transferred to a 15 mL conical tube, and pelleted at 400×g for 5 minutes. The supernatant was removed, and the cells were lysed by sonication (bioruptor) in 50 μL of EDTA-free lysis buffer at 4 °C for 10 minutes.

[0122] The lysate was clarified at 18,000×g for 15 minutes and analyzed by BCA assay. 30 μL of cell lysate was transferred to a 1.5 mL Eppendorf tube containing 30 μL of 7% TCA to precipitate the protein. The mixture was clarified at 18,000×g for 5 minutes, and 50 μL of the resulting solution was used for zinc detection assay.

[0123] Zinc concentration (nmol zinc / mg protein-free lysate):

[0124] 1 (PCC): 2.7 nmol / mg

[0125] 2 (PCC): 2.7 nmol / mg

[0126] 3 (PCC): 3.0 nmol / mg

[0127] 4 (HeLa): 2.3 nmol / mg

[0128] 5 (HeLa): 2.5 nmol / mg

[0129] 6 (HeLa): 2.3 nmol / mg

[0130] To achieve the various objects of the present invention, various embodiments of the present invention have been described. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Many modifications and adaptations thereof will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. A conjugate, the conjugate comprising: A transition metal catalyst coupled to a cell surface glycoprotein.

2. The conjugate according to claim 1, wherein the transition metal catalyst is coupled to the glycoprotein via a derivatized sialic acid linker.

3. The conjugate according to claim 2, wherein the transition metal catalyst and the derivatized sialic acid linker are coupled by click chemistry.

4. The conjugate according to claim 1, wherein the transition metal complex has the following formula: where M is a transition metal; where A, D, E, G, Y, and Z are independently selected from C and N; wherein R 3 to R 7 each independently represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of: alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 , and -R 9 OH, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; wherein R 1 is selected from the group consisting of a direct bond, an alkylene, an alkenylene, a cycloalkylene, a cycloalkenylene, an arylene, a heteroalkylene, a heteroalkenylene, a heterocycloalkylene, and a heteroarylene; where L is an optional linking moiety, the optional linking moiety selected from the group consisting of: amide, ester, sulfonamide, sulfonate, carbamate, and urea; and R 2 is selected from the group consisting of: alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic group, hydroxyl group, carboxyl group, halogen, alkoxy group, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , thiol, biotin, oxyamine and haloalkyl, wherein R 8 and R 9 are independently selected from the group consisting of: alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimide ester; and wherein X - is a counterion, and n is an integer from 0 to 20.

5. The conjugate according to claim 4, wherein R 2 is a click chemistry moiety selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide.

6. The conjugate according to claim 1, wherein the M is a platinum group metal.

7. The conjugate according to claim 6, wherein the M is iridium.

8. The conjugate according to claim 1, wherein the transition metal catalyst is a photocatalyst.

9. The conjugate according to claim 1, wherein the transition metal catalyst has an electronic structure for transferring energy to a protein labeling reagent.

10. The conjugate according to claim 9, wherein the energy transfer is Dexter energy transfer.

11. A system for analyzing the local microenvironment of a sialylated proteome, the system comprising: A protein labeling reagent; and A conjugate, the conjugate comprising a transition metal catalyst coupled to a cell surface glycoprotein via a derivatized sialic acid linker, wherein the transition metal catalyst has an electronic structure that allows energy transfer to the protein labeling reagent to provide a reactive intermediate.

12. The system according to claim 11, wherein the reactive intermediate is operative to label proteins within a predetermined radius of the conjugate.

13. The system according to claim 11, wherein the transition metal catalyst and the derivatized sialic acid linker are coupled by click chemistry.

14. The system according to claim 11, wherein the transition metal complex has the following formula: where M is a transition metal; where A, D, E, G, Y, and Z are independently selected from C and N; wherein R 3 to R 7 each independently represents one to four optional ring substituents, each of said one to four optional ring substituents being independently selected from the group consisting of: alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 , and -R 9 OH, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; wherein R 1 is selected from the group consisting of a direct bond, an alkylene, an alkenylene, a cycloalkylene, a cycloalkenylene, an arylene, a heteroalkylene, a heteroalkenylene, a heterocycloalkylene, and a heteroarylene; where L is an optional linking moiety, the optional linking moiety selected from the group consisting of: amide, ester, sulfonamide, sulfonate, carbamate, and urea; and R 2 is selected from the group consisting of: alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic group, hydroxyl, carboxyl, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , thiol, biotin, oxyamine, and haloalkyl, wherein R 8 and R 9 are independently selected from the group consisting of: alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimide ester; and wherein X - is a counterion, and n is an integer from 0 to 20.

15. The system according to claim 14, wherein R 2 is a click chemistry moiety selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide.

16. The system according to claim 14, wherein the M is a platinum group metal.

17. The system according to claim 16, wherein the M is iridium.

18. The system according to claim 11, wherein the transition metal catalyst is a photocatalyst.

19. The system according to claim 11, wherein the energy transfer is Dexter energy transfer.

20. The system according to claim 11, wherein the protein labeling reagent is bisaziridine.

21. The system according to claim 20, wherein the bisaziridine comprises a molecular marker.

22. A method for analyzing the local microenvironment of a sialylated proteome, the method comprising: Form a conjugate comprising a transition metal catalyst coupled to a cell surface glycoprotein via a derivatized sialic acid linker; Use the transition metal catalyst to activate a protein labeling reagent to a reactive intermediate; And Couple the reactive intermediate to a protein within a predetermined radius of the conjugate.

23. The method according to claim 22, wherein activating the protein labeling reagent to the reactive intermediate comprises energy transfer from the transition metal catalyst to the protein labeling reagent.

24. The method according to claim 22, wherein the protein labeling reagent is a bisaziridine.

25. The method according to claim 24, wherein the bisaziridine is functionalized with a marker.

26. The method according to claim 22, wherein the predetermined radius is 2 to 100 nm.

27. The method according to claim 22, wherein the reactive intermediate is quenched outside the predetermined radius, thereby excluding binding to biomolecules outside the predetermined radius.

28. The method according to claim 22, further comprising detecting or analyzing the protein coupled to the reactive intermediate.

29. The method according to claim 22, wherein the transition metal complex has the following formula: wherein M is a transition metal; wherein A, D, E, G, Y and Z are independently selected from C and N; wherein R 3 to R 7 each independently represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of: alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - , -C(O)OR 8 , and -R 9 OH, wherein R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; wherein R 1 is selected from the group consisting of a direct bond, an alkylene, an alkenylene, a cycloalkylene, a cycloalkenylene, an arylene, a heteroalkylene, a heteroalkenylene, a heterocycloalkylene, and a heteroarylene; wherein L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate and urea; and R 2 is selected from the group consisting of: alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic group, hydroxyl, carboxyl, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 、-OS(O2)R 9 、thiol, biotin, oxyamine and haloalkyl, wherein R 8 and R 9 are independently selected from the group consisting of: alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimide ester; and wherein X - is a counterion, and n is an integer from 0 to 20.

30. The method according to claim 29, wherein R 2 is a click chemistry moiety selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide.

31. The method according to claim 29, wherein the M is a platinum group metal.

32. The method according to claim 31, wherein the M is iridium.

33. The method according to claim 22, wherein the transition metal catalyst is a photocatalyst.

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